G-protein-mediated signaling underlies the sensing of extracellular stimuli in all eukaryotic organisms. G proteins, which are heterotrimers of Gα, Gβ, and Gγ subunits, couple GPCRs to effector proteins such as phospholipase C and to intracellular second messengers such as IP3. (Bourne, H. R., “How Receptors Talk to Trimeric G Proteins,” Curr. Opin. Cell Biol., 9:134–142 (1997)). Activated GPCRs promote the displacement of bound GDP by GTP on the surface of the Gα subunit and subsequent dissociation of the Gα subunit from the Gβ and Gγ subunits. Effector proteins are activated by binding to dissociated GTP-bound Gα subunits and/or by binding to Gβ and Gγ subunits.
Members of the Gαq class of G proteins couple GPCRs to phospholipase C, IP3 synthesis, and calcium release from intracellular stores. The development of robust assays for calcium fluxes in living cells, such as those that make use of fluorescent calcium dyes or calcium-dependent reporter genes, has facilitated the functional characterization of many GPCRs that couple to members of this class of G proteins. Moreover, many GPCRs that do not normally couple to Gαq-class G proteins can be linked to phospholipase C in two different ways: (1) using rodent Gα15 (or its human counterpart Gα16), a member of the Gαq class that couples to a large number of GPCRs in heterologous cells (Offermanns, S. and Simon, M. I., “G Alpha 15 and G alpha 16 Couple a Wide Variety of Receptors to Phospholipase C,” J. Biol. Chem., 270:15175–15180 (1995)); (2) using hyperactive Gαq variants (See U.S. Ser. No. 09/984,292 filed Oct. 29, 2001, and U.S. Provisional 60/293,770 filed Oct. 30, 2000 by Yong Yao et al., both incorporated by reference in their entirety herein), or Gαq chimeras that incorporate C-terminal coupling determinants from members of other classes of G proteins (Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D. and Bourne, H. R., “Substitution of Three Amino Acids Switches Receptor Specificity of Gq Alpha to that of Gi Alpha,” Nature, 363:274–276 (1993)). Recently, these two strategies have been combined to produce Gα16 chimeras that incorporate C-terminal coupling determinants from Gαz, a member of the Gαi class of G proteins; such Gα16/z chimeras have been shown to couple to several Gαi-coupled receptors that do not couple to Gα16 (Mody, S. M., Ho, M. K., Joshi, S. A. and Wong, Y. H. “Incorporation of G Alpha(z)-Specific Sequence at the Carboxyl Terminus Increases the Promiscuity of G Alpha(16) Toward G(i)-Coupled Receptors,” Mol. Pharmacol., 57:13–23 (2000)).
As described in the examples below, we have found, however, that the GPCRs that mediate sweet taste and umami taste (the savor of monosodium glutamate) in rats do not couple to Gα15, Gα16, or a Gα16/z chimera. Consequently, we have generated a series of Gα15 chimeras in which the 5-residue C-terminal tail of Gα15 was replaced by the tails of all other G proteins, and have used these chimeras to develop assays for the rat sweet and umami taste receptors. This invention relates to these Gα15 chimeras and their use in functional assays for mammalian taste receptors and other GPCRs that do not couple efficiently to Gα15.